Endothelium plays a central role in vascular physiology and pathology, maintaining vascular integrity and governing vascular permeability and smooth muscle tone. Endothelial function is regulated by both neurohumoral and mechanical stimuli. Neurohumoral stimulation occurs through classic receptor-mediated pathways, but the response to mechanical stimuli is poorly understood. An early event in the response of endothelial cells to mechanical and neurohumoral stimulation is activation of ion transporters, the physiologic significance of which is unknown. This laboratory has found that ion transporters in endothelial cells are also activated by changes in cell volume and are responsible for restoring cell volume. We believe that these transporters are activated via mechanical effects of changing cell volume, so that volume-sensitive ion transport provides a convenient system for studying mechanotransduction in endothelial cells. We propose to study volume-regulatory ion transporters in cultured bovine aortic endothelial cells to determine how they are regulated. Na-K-2Cl cotransport and cation channels will be measured by 86Rb fluxes, ion contents, and patch-clamp analysis. Regulation of protein phosphorylation by changes in cell volume will be examined with protein kinases, kinase activators and inhibitors in isolated cytoskeletons and intact cells. The phosphorylation of two proteins specifically in response to cell shrinkage will be used to probe for volume-sensitive kinases. The Na-K-2Cl cotransporter will be identified and its phosphorylation determined using 3H-bumetanide binding and WGA affinity chromatography. Regulation by Ca and Cl ions will be examined through manipulation of intracellular levels using ionophores and ion substitutions and through measurements of intracellular levels using fluorescent dyes. The results will provide important information about how endothelial cells regulate their volume and how they sense and respond to mechanical stress. Understanding how endothelial cells deal with mechanical stress will provide important insight into vascular diseases associated with endothelial injury such as atherosclerosis.